In the past two decades, fluorescent proteins (FPs) and FP?based biosensors have revolutionized biology. Notwithstanding this exponential rate of development and the abundant variety of FPs and FP?based biosensors, FP probes still lack desired essential properties for many biological and biomedical applications. This project employs a genetic code expansion technology and directed protein evolution to expand the capability of FPs to selectively detect chemical entities of biological significance, and to generate biological effectors with spatiotemporal control in biological systems. The following two specific aims are pursued: 1. Engineer genetically encoded fluorescence probes for detection of nanomolar H2S. 2. Develop unnatural fluorescent probes for light-induced 1O2 generation. The expected outcomes of this work will be the development of unique and useful research tools, including innovative fluorescent probes and optogenetic actuators. In particular, the proposed fluorescent sensors for H2S are expected to achieve ratiometric measurement, and improve detection limits by several hundred folds, compared to current fluorescent H2S probes. This will accelerate our understanding of how H2S acts as a modulator in cardiovascular, neuronal, immune, endocrine, and gastrointestinal systems. Moreover, novel genetically encoded photosensitizers will find broad applications for interrogation of cellular redox signaling pathways, for chromophore-assisted light inactivation (CALI), and for light?induced cell klling and photodynamic therapy. The impact of this project will extend beyond the biology of H2S and 1O2, because a general new strategy will be established for diversifying fluorescent probes and optogenetic actuators.